Assay timed by electrical resistance change and test strip

ABSTRACT

Assays for liquid analytes are performed on a bibulous matrix containing dried reagents which produce a visibly detectable reaction product. Application of liquid sample to the bibulous matrix is detected by measuring a drop in resistance across the matrix. A preferred test article for performing the method includes the matrix and a pair of spaced-apart electrodes in electrical contact with a reaction zone on the matrix. The test article is used in combination with a detection unit having means for probing the electrodes to determine when electrical resistance in the matrix has decreased. The assay methods and apparatus are particularly useful for performing enzyme assays where signal developed as a function of time is critical.

This invention was made with government support under SBIR Grant No. 1R43 HL48978-01 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods and apparatus fordetecting analytes in liquid samples. More particularly, the presentinvention relates to assays for detecting enzymes and components ofenzyme pathways where both the time and temperature of the assay arecontrolled.

Enzymes and enzymatic pathways play an important role in medicine andare the subject of many clinical tests. Examples of tests for singleenzymes include tests for amylase, creatine kinase, alanineaminotransferase, aspartate aminotransferase, streptokinase, andthrombin. Examples of tests for enzymatic pathways include theprothrombin time test, and the activated partial thromboplastin timetest. These later tests measure the enzymatic pathways involved in theextrinsic and intrinsic blood coagulation systems.

Tests involving enzymatic reactions tend to be technically demanding.Enzymes and enzymatic pathways are typically assayed by measuring therate at which the enzyme or enzymatic pathway in question converts aparticular enzymatic substrate into its product. Such rate measurementsrequire precise test timing since timing errors are directly translatedinto errors in the calculated amount of enzyme or component in an enzymepathway.

Temperature control is also critical since most enzymes have reactionrates that change dramatically as a function of temperature. Typically,higher temperatures produce a higher reaction rate, and lowertemperatures produce a lower reaction rate. Enzymatic pathways,consisting of a number of temperature sensitive enzymatic steps, areoften extremely temperature sensitive as a result of the cascade effect.

Because of these technical demands, most enzyme and enzymatic pathwaytests have tended to be complex, with performance generally limited toclinical laboratories. While such centralized testing may be adequatefor infrequent, or routine medical conditions, visiting a doctor'soffice or a clinic on a regular basis for frequent or emergency medicaltests is less acceptable. Thus there exists a need for convenient andsimple tests, that can be performed by unskilled users for themeasurement of enzymes and enzyme pathways.

A variety of simplified "test-strip" assays have been developed to allowsemi-skilled and unskilled users to detect analytes, such as pregnancyhormones, cholesterol, and glucose in urine, blood, and other patientsamples. These test strip assays are most useful with non-enzymaticanalytes where detection does not vary with minor fluctuations in testtime or temperature. As previously discussed, enzymatic reactions areless tolerant, and require more precise control over these variables,generally rendering them unsuitable for use in the home or othernon-clinical environment.

one such test strip for performing blood glucose analysis, availablefrom LifeScan Inc., Milpitas, Calif., relies on applying a drop of bloodto a polyamide membrane having impregnated reagents which produce achromogenic reaction in response to the glucose level in applied blood.Simplified low-cost tests such as this are often referred to as "hometests", to designate the fact that they have achieved a price andsimplicity level that would allow widespread adoption innon-professional settings.

For these reasons, it would be desirable to provide simplified assays,test articles, and test systems for detecting problematic analytes, suchas enzymes and components of enzyme pathways in a variety of patientsamples, such as blood, urine, and the like. In particular, the testarticles and test systems should permit simplified assay protocols,preferably allowing for an automatic timing cycle which is initiated assoon as a sample is applied to a test article. The test articles andtest systems should optionally also facilitate providing precisetemperature control of a test region on the article, preferably withoutthe need to enclose the test article in a heated chamber or otherstructure which limits the user access. The assays, test articles, andtest systems should be readily usable with small sample volumes,particularly with small blood volumes such as a single blood drop. Thetest articles should further inhibit loss of the patient sample from thetest article by evaporation or other means, particularly when using verysmall sample voluanes. The test article and test system should stillfurther provide for monitoring of the presence of sample within the testarticle and be able to warn the user when excessive amounts of samplehave been lost. Such test articles and test systems should be both easyto manufacture and easy to use, preferably being producible atrelatively low costs.

2. Description of the Background Art

Assay devices which detect the presence of an analyte based on theenzymatic conversion of a substrate to a visible or detectable productwithin an absorptive matrix are described in U.S. Pat. Nos. 5,059,525;5,059,394; 4,256,693; 4,935,346; 3,791,933; and 3,663,374. Analyticalapparatus having means for detecting sample application are described inU.S. Pat. Nos. 5,049,487 and 4,420,566. The '487 patent describes atiming circuit which is triggered by detecting a change in reflectancecaused by wetting of a porous matrix. The '566 patent describes themeasurement of light absorbance to confirm that a liquid sample has beenapplied to a slide prior to analysis. Systems for controlling thetemperature of analytical test substrates are described in U.S. Pat.Nos. 4,720,372; 4,219,529; and 4,038,030. Analytical test substratescomprising ion selective electrodes are described in U.S. Pat. Nos.4,171,246 and 4,053,381.

SUMMARY OF THE INVENTION

According to the present invention, apparatus and assays are providedfor performing timed assays, particularly timed enzymatic assays undertemperature control. The apparatus includes both a test article whichreceives a liquid sample being tested and a detection unit whichreceives the test article and optically determines a change in the testarticle resulting from presence of analyte in the sample over time. Theobserved change can thus be related to the presence (and usually amount)of analyte present in the sample. Such an apparatus permits performanceof simplified assay protocols and formats where the application ofsample to the test article present in the detection unit initiates atiming cycle and where the observed changes in the test article can thenbe detected as a function of time relative to the application of sample.

The test article comprises a bibulous matrix having one or more driedreagents present therein. The reagents are selected to produce adetectable signal in the presence of an analyte in a liquid sampleapplied to the matrix. A pair of spaced-apart electrodes are disposed oneither side of a target location on the matrix so that application of aliquid sample to the target location will lower electrical resistancebetween the electrodes. In this way, initial sample application can bedetected by monitoring the resistance across the electrodes, with alowering of the resistance initiating a timing cycle in the detectionunit. Additionally, a subsequent rise in electrical resistance betweenthe electrodes may indicate that sample has evaporated or otherwise beenlost from the target location which can be a particular problem withrelatively lengthy test protocols, particularly when employing smallsample volumes.

The test article can further be designed to reduce evaporative loss ofliquid sample. The spaced-apart electrodes will preferably be configuredto leave only a narrow gap therebetween, and the remaining portions ofthe matrix surface(s) will be covered by other materials. Usually, atleast a portion of the remaining covering will be transparent to permitoptical assessment of the matrix of the test article during assayprotocols as described in more detail hereinafter. With such a design,only the narrow gap between electrodes (which defines a target locationfor receiving sample) will remain uncovered, thus permitting liquidsample application but preventing significant evaporation from thebibulous matrix thereafter.

A preferred construction for the test article of the present inventionwill comprise a relatively thin membrane defining the bibulous matrix, apair of spaced-apart metal foil strips defining the electrodes andcovering substantially the entire top surface of the membrane, and atransparent layer or support, such as a clear plastic strip, coveringthe bottom surface of the membrane. Such a test article permits sampleapplication on the top of the membrane through the gap between adjacentelectrodes and further permits optical viewing of the membrane throughthe transparent bottom.

The detection unit of the present invention will include a support stagefor receiving the test article so that a reaction zone on the testarticle is disposed at a viewing location within the detection unit. Thedetection unit further includes means for measuring the electricalresistance across the test article when the test article is in place onthe support stage. Usually, the resistance detecting means will includea pair of plates or probes which contact the bibulous matrix on eitherside of the reaction zone (which is located at or near the targetlocation), preferably comprising a pair of plates which contact theelectrodes described above.

The detection unit, however, could be designed to function with testarticles which do not include discrete electrodes. In particular, thedetection unit could be designed to directly probe spaced-apartlocations on the membrane surface. While such a design will generally beless preferred, it is considered to be within the broad scope of thepresent invention.

The detection unit will further comprise a heater for heating the testarticle approximate the reaction zone. In the exemplary embodiment, theheater will include heated plates which contact the metal foilelectrodes on either side of the target location on the test article.The metal foil electrodes are thus able to transfer heat from the heatedmetal plates of the detection unit directly to the target location andreaction zone without blocking or obscuring the target zone for a sampleapplication.

The detection unit still further includes optical viewing means fordetecting an optical change in the reaction zone, typically through thetransparent layer of the test article. In exemplary embodiments, theoptical viewing means comprises a light source which directs lightagainst the reaction zone and an optical detector which detects lightemitted or reflected from the reaction zone.

According to the method of the present invention, a volume of a liquidsample is applied to the target location on a bibulous matrix, where thebibulous matrix comprises one or more dried reagents which in thepresence of analyte initiate time-dependent production of a detectablesignal. The precise time the sample is applied can be determined bymeasuring a change in electrical resistance across the target locationon the matrix. Production of the detectable signal is then measured atone or more times after the resistance change is first detected. In thisway, the timed-dependant production of the detectable signal can becarefully monitored over time and accurately related back to thepresence in amount of analyte in the liquid sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded drawing of an exemplary embodiment of a testarticle constructed in accordance with the principles of the presentinvention.

FIG. 2 is a schematic representation showing the test article in FIG. 1present in a detection unit.

FIG. 3 is an exemplary graph showing the range in electrical and opticalproperties of a test article upon contact with analyte in a liquidsample.

FIG. 4 illustrates the test article and detection unit utilized in theexamples of the Experimental section.

FIG. 5 is the result from an experiment using the test article anddetection unit shown in FIG. 4. In this experiment, the results from 27blood samples with varying prothrombin times were compared withprothrombin times obtained from a standard reference system.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Assays according to the present invention are useful for detecting awide variety of soluble analytes in virtually any type of biological orother sample which is liquid or which can be liquified. The methods andapparatus will find their greatest use with patient specimens, such asblood, serum, plasma, urine, cerebral fluid, spinal fluid, ocular lensliquid (tears), saliva, sputum, semen, cervical mucus, scrapings, swabsamples, and the like, but might also find use with food, environmental,industrial, and other samples where substances can be detected usingenzymatic, immunological, and similar techniques. The methods andapparatus of the present invention are particularly useful for detectinganalytes in very small liquid samples, typically having a volume in therange from about 3 μl to 50 μl, usually from 5 μl to 30 μl, but willalso be suitable for use with much larger samples. In particular, themethods and apparatus of the present invention will be useful foranalyzing very small blood samples, typically comprising a single dropof blood, employing non-skilled or semi-skilled personnel, frequentlybeing self-administered.

The methods and apparatus of the present invention are also particularlysuited for perfoming enzymatic assays within a bibulous matrix where ananalyte in a sample applied to the matrix initiates, modulates, orotherwise affects the enzymatic conversion of a substrate (initiallydried within the matrix) into an optically detectable product, such as acolored, luminescent, or fluorescent product. The present inventionprovides for very accurate timing of such enzymatic reactions based oninitiation of a timing cycle at the moment the liquid sample isinitially applied to the test article. The test article and detectionunit can further provide for accurate temperature control of the testarticle, further assuring accuracy of the test result.

Assays according to the present invention provide for sensing of sampleaddition to a test article by measuring electrical resistance across aportion of the bibulous matrix to which the sample is applied. As thebibulous matrix and all reagents present therein are initially in a drystate, the resistance of the bibulous matrix prior to sample additionwill be relatively high. Application of a liquid sample will immediatelylower the electrical resistance across that portion of the matrix towhich it is applied, thus providing a marker or trigger for initiationof a timing cycle. In the case of enzymatic reactions, build-up of theoptically detectable product will take some time and is usually highlydependent on the amount of analyte present within the sample (as well asother factors such as temperature). By providing control circuitrywithin or in combination with the detection unit, the amount ofoptically detectable product accumulating within the test article overtime can be precisely determined. Based on one or more such data points,usually a plurality of such data points, the amount of analyte initiallypresent in the sample can then be accurately calculated.

The detection unit of the present invention will comprise means formonitoring and measuring resistance across that portion of the testarticle to which liquid sample is to be applied. The resistancedetecting circuitry will be connected to timing circuitry, which in turnwill be connected to a suitable calculating means. Conveniently, thedetection unit may include an interface for connecting to a digitalcontroller, usually a microprocessor, which may be in the form of ageneral purpose personal computer. In this way, much of the timing andanalytical function of the system of the present invention can beperformed within the digital controller, with the specialized detectionand sample conditioning functions being performed by the specializedapparatus of the present invention.

Referring now to FIG. 1, an exemplary test article 10 constructed inaccordance with the principles of the present invention will bedescribed. The test article 10 includes a bibulous matrix structure 12,typically in the form of a flat membrane; a support structure 14,typically in the form of a transparent strip; and an electrode structure16, typically comprising a pair of spaced-apart electrodes 18 separatedby a gap 20. The membrane 12, support structure 14, and electrodestructure 16 will be laminated together, for example by adhesive layers22 and 24, as illustrated. The adhesive layers 22 and 24 will includecentral apertures 26 and 28 which permit viewing of the membrane 12through the support structure 14 and application of sample to themembrane through the gap 20 in electrode structure 16. Preferably, theelectrode structure 16 will include additional slots 30 formed in theelectrodes 18, which slots define a target location for the applicationof liquid sample on the test article 10.

The bibulous matrix 12 will be composed of a material which can absorbliquid and which can contain, in dried form, the reagent(s) necessaryfor performing a desired assay. A wide variety of bibulous matrixmaterials might be used, including paper, methyl cellulose, porouspolymers, and the like. The dimensions of bibulous matrix 12 should besuch that at least a portion of the matrix can be saturated with liquidsample to both solubilize the necessary reagent(s) and to permittransport of the detectable reaction product to the lower side of thematrix so that it will be visible through the support structure 14.

In the preferred embodiment where small samples of blood are beinganalyzed, the bibulous matrix 12 will be a porous membrane structurecomposed of a hydrophilic (bibulous), non-swellable polymeric matrixmaterial having pore dimensions which permit entry of blood plasma andproteins while excluding blood cells, particularly red blood cells(erythrocytes). The membrane should be composed of a single, continuouspolymeric material with a foam-like structure consisting of a torturousnetwork of channels having widths on the order of microns (μm). Thetorturous network of channels is "densely packed" in that the "voidvolume" occupied by the empty space of the channels is an appreciablepercentage of the total membrane volume, typically 10% or greater. Sinceall reaction chemistry, and subsequent signal generation, takes place inthe void volume, a high void volume is desirable for producing a strongsignal. A torturous network of channels is desired over straight anddirect pores, (such as the short, direct pores obtained with nucleoporemembranes), as longer average channel lengths tend to produce anincreasing isolation between the zone of the membrane where reactionchemistry is occurring, and the excess sample remaining on the surfaceof the membrane. This helps to render the system less sensitive tovariations in applied sample volume.

In the specific case of blood coagulation assays, the porous membranestructure 12 will be impregnated with reagents necessary to inducecoagulation in blood plasma which enters the interior of the porousmatrix and to produce a detectable signal as an indication of thecoagulation capability of the blood. It is particularly critical to thepresent invention that the polymeric matrix material of the porousmembrane 12 be substantially free from interference with the coagulationpathway which is being induced. In particular, the polymeric matrixmaterial should be free from surface effects, interactions, andartifacts which might induce coagulation or inactivate components suchas enzymes, of the initiated pathway. Unintended initiation of acoagulation pathway could lead to false positive determinations whileenzyme inactivation could lead to false negative determinations. It istherefore important that the polymeric matrix material have no promotingor diminishing effect on the coagulation reactions occurring within themembrane. Criteria can be for determining if a membrane is acceptablefor use in coagulation testing are set forth in detail in copendingapplication Ser. No. 07/874,667, the full disclosure of which isincorporated herein by reference. A particularly preferred polymericmatrix material for performing blood coagulation assays is a 0.45 μmasymmetric polysulfone membrane material available fromFilterite-Memtec, 9690 Deeveco Road, Suite 7, Timonium, Md. 21093,Catalog No. BTS-25.

The region of bibulous matrix 12 which is beneath the sample applicationtarget location slots 30 will be the reaction zone. It is in this regionthat the matrix 12 is first wetted and where the actual chemicalreaction which results in production of a detectable reaction productoccurs. When used with very small samples, as described above, thereaction zone will typically be relatively small, frequently being 1 cmin diameter or less, often being less than 0.5 cm. The remaining regionsof the matrix 12 which are not wetted by the sample will not undergo achemical reaction and will not accumulate visible reaction product.

Chemical reagents necessary for performing an assay according to thepresent invention will be impregnated within the bibulous matrix 12 andwill be reconstituted by application of the liquid sample thereto. Forthe preferred enzymatic assays of the present invention, the reagentswill include an enzyme substrate which is converted into an opticallydetectable product, typically a fluorescent, luminescent, or coloredproduct, as a result of interaction with an enzyme. The enzyme may bethe desired analyte or related to the analyte, or the enzyme may beadded to the liquid sample and the production of detectable product bythe enzyme modulated or otherwise affected by presence of analyte in thesample. The substrate may be a natural enzyme substrate which produces anatural detectable product, e.g. in the case of peroxidases, oxidases,hydrolases, and the like, or may be a synthetic substrate comprising asubstrate group, such as a polysaccharide or peptide, which is cleavablylinked to a reporter molecule, such as a chromogenic, chemiluminescent,or fluorogenic molecule. The presence or activity of the enzyme in thesample results in cleavage of the linker, causing a change in theoptical characteristics of the reporter molecule. A variety of usefulsubstrates are described in Haughland, Molecular Probes Handbook ofFluorescent Probes in Research Chemicals, Molecular Probes, Inc.,Eugene, Oreg., the full disclosure of which is incorporated herein byreference.

In the exemplary case of blood coagulation assays, necessary reagentsinclude a coagulation initiator which initiates a preselected event orstage in either an extrinsic or intrinsic coagulation pathway and asubstrate which is activated by a component which is produced in asubsequent stage of the coagulation pathway. A buffer will also beprovided to maintain the test pH within a range compatible with thecoagulation pathway, and optional reagents include flow control agentswhich decrease the chromatographic separation of the various testcomponents as blood plasma enters the membrane, cofactors which sustainor enhance the chemical reactions of the coagulation pathway, stabilityenhancers, and pigments which enhance the optical characteristics of thetest article. Typically, these reagents will be combined in one or moreaqueous solution(s) which are applied to all or a portion of thepolymeric matrix material. The matrix material may then be dried orlyophilized (and optionally mounted on the handle 14) to form a testarticle having the reagents non-covalently adsorbed therein. In somecases, it may be possible to covalently attach at least some of thereagents, although covalent attachment will usually not be necessary.Particular coagulation inhibitors, substrates, buffers, coagulationcofactors, and fluid control agents are set forth in application Ser.No. 07/874,667, the full disclosure of which has previously beenincorporated herein by reference.

The support structure 14 may take a variety of forms. The supportstructure 14 is intended primarily to act as a physical support for theremaining components of the test article 10 and should be opticallytransmissive, preferably being completely transparent at the lightwavelengths of interest to the assay protocol. In a preferred aspect ofthe present invention, the support structure 14 will also act as amoisture barrier in preventing loss of sample from the bibulous membrane12. Suitable support structures 14 may be composed of transparentplastics, such as polystyrene, which is sufficiently thick and rigid toserve as a handle to permit manipulation of the test article 10 in themethod steps of the present invention. Polystyrene strips having alength in the range from about 2 cm to 10 cm, a width in the range fromabout 0.5 cm to 2 cm, and a thickness in the range from about 0.1 mm to0.5 mm have been found to be acceptable.

The electrode structure 16, is intended primarily to facilitatemeasurement of the electrical resistance across the reaction zone ofbibulous matrix 12. It will be appreciated that the methods of thepresent invention could utilize electrical resistance probes whichcontact the bibulous matrix directly, that is without the need forproviding intermediate contacting electrodes. The preferred test article10 of the present invention, however, provides a suitable electrodestructure in order to facilitate electrical resistance measurement usingthe detection unit, as described in more detail hereinafter.

The electrode structure 16 preferably also serves as a cover for thebibulous matrix 12 to inhibit evaporative and other losses of samplefluid therefrom. Thus, the electrode structure 16 will generally cover amajority of the exposed surface of the bibulous matrix 12, typicallyleaving only the small gap 20 and slots 30 therebetween.

Conveniently, both electrode halves 18 of the electrode structure 16will be composed entirely of a conductive material, usually a metalfoil. Use of the metal foil also acts to enhance heat transfer to thereaction zone of bibulous matrix 12, as described in more detail inconnection with the detection unit hereinafter. It will be appreciated,however, that the individual electrodes 18 need not be composed entirelyof electrically conductive material, and in fact only need to define adiscrete conductive path from the bibulous matrix 12 to a location whichcan be probed or connected by the detection unit.

It should be further appreciated that the test articles of the presentinvention are not limited to structures having the electrode 16 andsupport structure 14 sandwiched about a bibulous matrix 12. Testarticles according to the present invention require only that a pair ofspaced-apart electrodes be provided on either side of a sample targetlocation on the bibulous matrix, where the electrodes facilitateinterconnection with a electrical resistance measuring mechanism, suchas that provided by the detection unit of the present invention.

The adhesive layers 22, 24 may be composed of any suitable material,typically being double sided tape, such as that available from 3MCorporation, Minneapolis, Minn..

Referring now to FIG. 2, a detection unit 32 is schematicallyillustrated. The detection unit 32 includes plates 34 which contact theelectrodes 18 of the test article 10 when the test article has beeninserted into the detection unit. The contact plates 34 act as probes inmeasuring electrical resistance across the reaction zone in matrix 12beneath the target location slots 30. Plates 34 will be connected toconventional electrical resistance monitoring circuitry in order toprovide an output suitable for a digital control unit (not illustrated)or microprocessor.

In a preferred aspect of the present invention, plates 34 will beheated, typically by conventional electrical resistance heaters. Forexample, the plates 34 may have heating coils on the surface thereof orembedded therein. The heating coils, in turn, could be connected to aconventional power supply 35, with heat being controlled by conventionalcontrol circuitry or by a separate computer or other digital controller.Heat provided by plates 34 will be transferred by metallic electrodeplates 18 to the matrix 12 to an area at least partially overlappingwith the reaction zone.

Detection unit 32 further includes a system 40 for optically viewing thereaction zone when the test article 10 is in place within the unit. Thisoptical system may monitor reflectance, fluorescence, or luminescence.In this example, a fluorescence system 40 is illustrated. Thisfluoresence system 40, as illustrated, includes a light source 42 and anotch filter 44 which together provide a light beam 46 which falls onthe reaction zone of test article 10 through the transparent supportstructure 14. Reflected or emitted light 48 passes through a secondnotch filter 50, with filtered light being detected by photodetector 52.In the exemplary case of blood coagulation assays, detection willusually be based on fluorescence, where the light beam 46 is provided atan excitation wavelength and the light beam 48 is emitted at a knownemission wavelength.

In summary, an assay according to the present invention may be performedby applying a liquid sample through the target location 30 on theelectrode structure 16 of the test article 10. The liquid sample willflow through the gap 20 and other slots in the target location into areaction zone within bibulous matrix 12. The presence of analyte in thesample will affect or modulate the production of a detectable reactionproduct within the matrix 12, eventually providing an opticallydetectable change on the lower surface of the matrix. The production ofoptically active reaction products may be observed by directing a lightbeam 46 through the transparent support 14 onto the reaction zone ofmembrane 12. The fluorescent material at present, will emit light at afluorescent wavelength which is eventually detected by a photodetector52. The amount of signal produced over time will depend on the amount ofanalyte present in the sample.

FIG. 3 illustrates the changes in resistance and optical characteristicsthat typically take place during the course of an assay. Before theapplication of a fluid sample, the resistance across the electrode gapis extremely high. By contrast, the optical signal (here shown asfluorescence) is low. Upon application of sample at time zero, there isan immediate drop in resistance. By contrast, there is no correspondingchange in the optical signal until appreciable amounts of the enzymesubstrate have been converted to detectable product within the reactionmembrane. As time continues, the resistance across the electrodejunction on the reagent strip may increase, due to drying of the strip.As previously discussed, the system may choose to reject a particularsample as being insufficiently moist if the resistance measurementbecomes too high.

The following examples are offered by way of illustration, not by way oflimitation.

EXPERIMENTAL General Methodology

Instrument:

Observations were performed using a prototype instrument. The instrumentoptics included a Siemens BPW-34B photodetector mounted below a 550nanometer filter with a 25 nanometer bandwidth (S25-550-A, CorionCorporation, Holliston, Mass.). The specimens were illuminated by aMini-Maglite^(TM) LM3A001 light bulb (Mag Instrument Inc., Ontario, CA)with output filtered through a 500 nanometer filter with a 25 nanometerbandwidth (Corion Corporation, S25-500-A). The output from thephotodetector was amplified by an instrumentation amplifier (describedon page 89 of the IC Users Casebook, 1988, by Joseph Car, Howard Samms &Company), digitized by a 12 bit analog to digital converter, andrecorded on an IBM compatible personal computer. Temperature control wasachieved by placing reagent strip 10 in a heated reagent stage 50, asshown in FIG. 4. The stage 50 included an upper heater and a lowerheater with test strip 10 in the middle. A slot 52 was provided into theupper stage to facilitate insertion and removal of the strip 10 into thestage, and to provide easy sample access from the top. The stage washeated by eight, 200 ohm 1/4 watt resistors connected in parallel. Forthe lower heater, four resistors were mounted on a thin circuit board 54and were used to heat a 0.015" thick aluminum plate 56, with a 0.5"diameter hole 58 as an optics aperture. For the upper heater, fourresistors were mounted on a circuit board 60 and used to heat twoseparate, and electrically isolated, 0.015" thick aluminum plates 62.Circuit board 60 additionally had a 0.45" by 1.5" opening 52 in it toallow easy application of sample to the reagent strip's aperture 30.

As shown in both FIG. 2 and FIG. 4, aluminum plates 62 on the stage'supper heater made a tight contact with the opposite sides of the teststrip's foil surface electrodes. The stage's relatively thick aluminumplates acted to evenly distribute the heat emanating from the resistors,and helped insure good heat flow to the test strip's foil surface. Thetight contact between the meter's aluminum plates, and the test strip'sfoil electrodes, was also used to form an electrical circuit that wasused to detect the state of the strip's fluid detection sensor.

The temperature was regulated by monitoring the stage via an AcculexRTDR-2 temperature sensitive resistor (Keithley MetraByte InstrumentsCorp., Taunton Mass.). The electronics used to construct the fluidsensor and the temperature sensor are standard resistance monitoringcircuits described on pages 55 and 62 of the Dascon-1 Manual, copyright1983, by Metrabyte Corporation, Taunton, Mass. Unless otherwise noted,temperatures were maintained at 37° C. by a feedback control programmonitored by an IBM compatible personal computer. The computer systemcontrolled a switching circuit that energized the heater, using a 6 voltpower source, whenever temperatures dropped below 37° C. In use, reagentstrips were allowed to pre-equilibrate to 37° C. for a minimum of 60seconds before sample was applied.

Preparation of Boc-Val-Pro-Arg-Rhodamine 110:

Five grams of Boc-Val-Pro-OH (catalog A-2480) were purchased from SachemBioscience, Inc., Philadelphia Pa.. This was conjugated onto (CBZ-Arg)2-Rhodamine-110 to produce (Boc-Val-Pro-Arg) 2-Rhodamine 110 followingthe methods of Mangel, et. al. in U.S. Pat. No. 4,557,862 and U.S. Pat.No. 4,640,893. This formed the enzyme substrate used to detect thrombinproduction in the exemplary prothrombin time assay disclosed here.

Preparation of photo-etched electrode apertures:

A etching pattern was prepared, consisting of a repeated series of thefollowing configuration: one 0.625" long, 0.015" wide electrode gap, asshown as 20 on FIG. 1, and two 0.185" long, 0.007 wide fluid aperturesshown as 30 on FIG. 1, arranged in a cross configuration on the centerof the electrode gap, with each slit separated by an angle of 60° fromthe other slits. This pattern was etched onto I mil thick aluminum foilby Accutech, Inc., San Fernando, Calif. using conventional photo-etchingtechniques. The apertures were trimmed to a square 0.500"×0.500"configuration, with the aperture centered in the middle of this square.

Preparation of coated membrane:

Coated membranes were prepared generally as described in copendingapplication Ser. No. 07/874,667, now abandoned. In a 20 ml vial, using asmall magnetic stirrer, the following were combined: 6 ml 0.2M HEPES pH7.4; 2 ml H2O; 1 ml 100 mM CaC12; and 500 mg Sigma poly vinyl alcoholP-8136; and were stirred for about 2 hours until the PVA had totallydissolved.

One gram of Sigma protease-free bovine serum albumin A-3292 was added,and the mixture was allowed to stir for about 20 minutes until it hadtotally dissolved. A solution of 4.5x concentrated Dade-C thromboplastin(Baxter B4216-20, Baxter Healthcare Corporation, Miami, Fla.), wasprepared by adding 880 μl H2O to nominal 4 ml containers ofthromboplastin. One ml of the concentrated (4.5x) Dade-C thromboplastinwas added to the dip and was stirred for 10 minutes. One ml of aconcentrated (4 mg/ml) solution of fluorescent thrombin substratedissolved in 50% isopropanol, 50% H20 was then added, and the resultingmixture stirred for 10 minutes.

Membrane dip:

The large pore side (dull side) of a BTS-25 0.45 μm asymmetricpolysulfone membrane (Memtec/Filterite Corporation, Timonium Md.) wasrapidly coated with fresh dip, and the excess dip was gently squeegeedoff. The coated membrane was immediately dried in a mechanicalconvection oven at 50° C. for 15 minutes. The membrane was then storedwith desiccant under cool (4° C.) conditions until use.

Preparation of membrane strips:

The reagent's support layer 14, FIG. 1, was composed of 10 mil thicktransparent styrene. The various layers were held together by 0.5" wide3M 415 double sided adhesive tape, made by the 3M Corporation. Tohand-assemble the strips, two preliminary assemblies were first created.The first assembly consisted of a series of 0.25" diameter holes spaced0.500" apart, pre-punched in the double sided adhesive tape. Thisadhesive tape 24, FIG. 1, was then applied to a length of foilcontaining a series of repeating apertures, each 0.500" apart. Excessfoil, used to keep the repeating aperture structures intact in theabsence of the adhesive tape, was then trimmed away, producing an"aperture tape" assembly with adhesive on one side, and a repeatingseries of electrode apertures on the other side. This was kept flat tokeep the delicate electrode aperture structures intact.

A second preliminary assembly was created consisting of a strip oftransparent styrene laminated with 3M 415 tape and a length of 0.5" widetreated reaction membrane. To avoid migration of adhesive into thereaction membrane, and it's possible deleterious effects on storage, aseries of 0.25" holes were punched every 0.500" in the sections of thetape that were immediately below the "reaction zone" on the final strip22, FIG. 1. Thus the coated membrane in the reaction zone did not comeinto immediate contact with any materials except the metallic foilaperture covering. The two preliminary assemblies were then aligned withthe aid of a light box, and laminated together. The repeating subunitson the final assembly were then cut into individual strips for testing.

This reagent, and the meter described previously, were then used toperform the following experiments.

Experiment 1:

Resistance drop upon addition of biological fluids: Reagent strips wereprepared according to the above methods. The resistance across thestrips was monitored as a function of time. Before addition of blood,the resistance across the electrode junction was effectively infinite(>100 mega ohms). Upon addition of blood, the resistance immediatelydropped to about 30 kilo ohms.

Experiment 2:

Detection of insufficient sample by resistance readings. Reagent stripeprepared according to above methods were each given 1.0, 2.0, 3.0, and4.0 μl of whole blood. After six minutes of reaction time, therespective resistance measurements of the various strips were:

    ______________________________________                                        Sample Size   Resistance                                                      ______________________________________                                        1.0 μl     2.5         mega ohms                                           2.0 μl     400K        kilo ohms                                           3.0 μl     60K         kilo ohms                                           4.0 μl     40K         kilo ohms                                           ______________________________________                                    

Insufficient sample thus could be detected by a substantial increase inresistance.

Experiment 3:

Effect of metal foil on reaction kinetics at low humidity: Strips wereprepared omitting the metallic foil, and studied with Sigma coagulationcontrol I, II and III plasma in a low ambient humidity environment andmanual initiation of test timing. In the absence of the surfacecovering, lower fluorescence intensity developed, and the reactionkinetics for level II and level III (prolonged and very prolonged PTtime controls) was significantly prolonged.

Experiment 4:

Effect of a hole in the transparent reagent support on reaction kineticsat low humidity: Strips were prepared containing the metallic foil, butadditionally with a 0.25" diameter hole placed in the transparentsupport immediately below the reaction zone on the membrane. These werestudied with coagulation control I, II and III plasma (C-7916, C-8916and C-9916, Sigma Chemical Company, St. Louis, Mo.) in a low ambienthumidity environment. In the absence of the surface covering, lowerfluorescence intensity developed, and the reaction kinetics for level IIand level III (prolonged and very prolonged PT time controls) wassignificantly prolonged.

Experiment 5:

Effect of metallic foil cover on temperature equilibration of reagent:Before they are applied to the meter, the test samples may be at avariety of different initial temperatures (typically 15°-30° C.). Togive accurate results, the test article must equilibrate these differentsamples to the same reaction temperature (typically 37° C.) as rapidlyas possible. This experiment measured the relative effectiveness of ametallic foil cover, versus a non-metallic plastic cover, at rapidlyequilibrating different samples.

In this experiment, a series of test articles were made up with either 1mil thick aluminum covers, or 5 mil thick transparent styrene covers.These were placed into a meter stage and equilibrated to 37° C. 10 μldrops of water (at 4° C.) were then applied, and the temperaturesmonitored by a non-contact infrared thermometer (C-1600 meter, LinearLaboratories Company, Fremont, Calif.). Both the aluminum foil and thetransparent styrene were painted flat black (Model 2529 marker, TestorCorp., Rockford Ill.), to provide equivalent conditions for the infraredbeam. The resulting temperatures were:

    ______________________________________                                        Time after application                                                        (sec.)           Foil temp.                                                                              Plastic temp.                                      ______________________________________                                         1               33        20                                                  5               35        23                                                 10               37        26                                                 15               37        28                                                 30               37        33                                                 45               37        37                                                 ______________________________________                                    

The results show that the foil covering equilibrated the sample to thedesired temperature more quickly than did the plastic covering.

Experiment 6:

Performing fluorescence assays without a meter hatch: In the followingexperiment, the meter was programmed to take fluorescence measurementsby turning its light source on briefly every 15 seconds, and recordingthe fluorescence value. Immediately before the meter's own light sourcewas turned on, however, the meter also recorded a "background" value.This background value was then subtracted from each fluorescencemeasurement.

It was found that when a non-light transmissive covering with a narrowslit was used, such as the metal foil arrangement discussed previously,the system was remarkably insensitive to the presence or absence ofexternal light--e.g. normal room lighting, or indirect sunlight. This isattributed to a combination of factors, including the great attenuationof ambient light by the metal foil covering on the strip, the highfluorescence efficiency and amounts of the Rhodamine 110 fluorophoreused in this experiment, and the background subtracting algorithm usedhere.

Experiment 7:

Clinical study: Venous blood from 27 patients, including 20 patientsbeing treated with Coumadin, was drawn and stored in citrate. 10 μlsamples of whole blood from each patient were then run on the device asdescribed above. Fluorescence measurements were taken every 15 secondsfor six minutes. The time required for the fluorescence intensity toreach half maximum (T50%)was plotted versus the reference prothrombintimes as determined on a MLA Electra 750 coagulation meter. A regressionanalysis was done using Microsoft Excel 4.0 software.

A best fit polynomial to relate the T50% levels to the referenceinstrument was found to be prothrombin time (corrected)=1.93+0.079*T50%. The correlation coefficient (R²) was 0.92. The results from thisexperiment are shown in FIG. 5. The results show that this configurationcan give good clinical results.

Although the foregoing invention has been described in some detail byway of illustration and example, for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. An assay for determining an analyte in a liquidsample, said assay comprising:applying a volume of the sample to atarget location on a bibulous matrix, wherein the matrix comprises oneor more dried reagents which in the presence of the analyte initiatetime-dependent production of an optically detectable signal within thematrix; measuring a change in electrical resistance across the targetlocation which results from the application of liquid sample; initiatinga timing cycle when a lowering of electrical resistance is firstmeasured; and measuring the production of said optically detectablesignal within the matrix at one or more times during the timing cycle.2. An assay as in claim 1 wherein the sample volume is from 3 μl to 50μl.
 3. An assay as in claim 1, wherein the sample is a patient sample.4. An assay as in claim 3, wherein the patient sample is blood.
 5. Anassay as in claim 1, wherein the analyte is an enzyme and the reagentsinclude an enzyme substrate which produces an optically detectablechange after exposure to the enzyme.
 6. An assay as in claim 5 whereinthe optically detectable change is fluorescence, luminescence, or colorchange.
 7. An assay as in claim 5, wherein the analyte is a component ofa coagulation pathway and the enzyme substrate produces the detectablesignal upon activation by said component.
 8. An assay as in claim 1,further comprising controlling the temperature of the bibulous matrix to37° C.
 9. An assay as in claim 1, further comprising continuouslymonitoring the electrical resistance across the target location, wherebyloss of liquid sample can be detected.
 10. An assay as in claim 1,wherein the production of signal is detected at a plurality of timepoints over a preselected time duration.
 11. An improved enzyme assay ofthe type wherein a liquid sample suspected of containing the enzyme isapplied to a bibulous matrix-containing reagents which result inproduction of an optically detectable signal over time, wherein theimprovement comprises measuring a change in electrical resistance withinthe matrix resulting from application of the liquid sample and opticallydetecting the production of signal at one or more times after theelectrical resistance change is first measured.
 12. An improved assay asin claim 11, wherein electrical resistance is measured by contacting apair of electrodes which are secured to the bibulous matrix on eitherside of a location where liquid sample is applied.
 13. An improved assayas in claim 11, wherein the electrical resistance is continuouslymonitored to assure that the liquid sample has not been lost during theassay procedure.
 14. An improved assay as in claim 11, wherein theproduction of signal is detected at a plurality of time points over apreselected time duration which commences when the change in electricalresistance is first measured.
 15. A test article comprising:a bibulousmatrix; one or more dried reagents which are present within the matrixand which, when wetted by a liquid sample, initiate an opticallydetectable chemical reaction with an analyte present in the sample; anda pair of spaced-apart electrodes proximate a sample target location ofthe matrix, wherein the electrodes are disposed so that application ofthe liquid sample to the target location will lower electricalresistance between the electrodes.
 16. A test article as in claim 15,wherein the spaced-apart electrodes are secured to the bibulous matrixand in electrical contact with at least a portion of a surface of thematrix on opposite sides of the target location.
 17. A test article asin claim 16, wherein the electrodes are metal foil strips which areattached to the matrix surface by an adhesive.
 18. A test article as inclaim 15, wherein the gap between the spaced-apart electrodes has awidth in the range from 0.1 mm to 2 mm, whereby the electrode inhibitsevaporation of sample from the matrix.
 19. A test article as in claim18, further comprising a transparent support element attached to asurface of the matrix opposite to the electrodes.
 20. A test article asin claim 15, wherein the bibulous matrix is composed of a hydrophilic,non-swellable material which is free from interference with acoagulation pathway.
 21. A test article as in claim 20, wherein thereagents comprise a coagulation initiator and a substrate which producesa detectable signal upon activation by a component of the coagulationpathway.
 22. A test article as in claim 21, wherein the coagulationpathway component is thrombin and the substrate is a peptide cleavablybound to a reporter molecule, wherein thrombin binds the peptide andcleaves the reporter molecule to produce the detectable signal.
 23. Atest article as in claim 22, wherein the detectable signal isfluorescence.
 24. A test article comprising:a bibulous matrix; one ormore dried reagents which are dispersed within the matrix and which,when wetted by a liquid sample, initiate an optically detectableenzymatic reaction with an enzyme analyte present in the sample; and apair of spaced-apart metal strips attached to a surface of the matrix todefine a sample target location therebetween, whereby application of theliquid sample to the target location will lower electrical resistancebetween the metal strips.
 25. A test article as in claim 24, where themetal strips are attached to the matrix surface by an adhesive.
 26. Atest article as in claim 24, wherein the gap between the spaced-apartmetal strips has a width in the range from 0.1 mm to 2 mm, whereby thestrips inhibit evaporation of sample from the matrix.
 27. A test articleas in claim 26, further comprising a transparent support elementattached to a surface of the membrane opposite to the metal strips. 28.A test article as in claim 24, wherein the bibulous matrix is composedof a hydrophilic, nonswellable material which is free from interferencewith a coagulation pathway.
 29. A test article as in claim 28, whereinthe reagents comprise a coagulation initiator and a substrate whichproduces a detectable signal upon activation by a component of thecoagulation pathway.
 30. A test article as in claim 29, wherein thecoagulation initiation is thrombin and the substrate is a peptidecleavably bound to a reporter molecule, wherein thrombin binds thepeptide and cleaves the reporter molecule to produce the detectablesignal.
 31. A test article as in claim 30, wherein the detectable signalis fluorescence.